Expression profiling based on histocultures

ABSTRACT

Methods of obtaining faithful expression libraries from tissue samples comprise extraction of RNA from intact tissue cultured in three-dimensional sponge-gel based histocultures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. Ser. No. 10/712,781 filed 12Nov. 2003, which claims benefit under 35 U.S.C. § 119(e) to U.S. Ser.No. 60/425,945 filed 12 Nov. 2002. The contents of these applicationsare incorporated herein by reference in their entirety.

TECHNICAL FIELD

The invention relates to preparation of samples, especially tumorsamples, as sources of mRNA for expression analysis.

BACKGROUND ART

There is an extensive history of the use of histocultured tumor samplesfor use in prognosis of tumor development and as a tool for predictingresponsiveness to drugs. These histochemical techniques, which are thebasis of histoculture drug response assay (HDRA™), have an extensiveliterature. The general features of this technique are described, forexample, in a recent paper by Singh, B., et al., Head and Neck (2002)24:437-442. As described in this paper, briefly, biopsied tissue iswashed and cut into 1 to 2 mm³ fragments and placed onto 0.5 cm² piecesof collagen sponge-gel (Gel Foam, Pharmacia & Upjohn, Inc.) in equalquantities. The sponge-gel cultures are then placed into DMEM/Ham's F12medium with 10% fetal calf serum and gentamicin (50 μg/ml). The culturesare then incubated for 24 hours at 37° C. and 5% CO₂. Modifications ofthis technique are also permissible, provided the three-dimensionalnature of the sample is preserved.

It has now been found that in addition to their usefulness as prognosticand drug-screening tools, such cultures are also useful as sources formessenger RNA as a substrate for expression profiling. This issignificant in view of the problems associated with providing reliableexpression libraries, in particular when derived from patient sampleswhere extraction of high-quality, non-degraded RNA is difficult in viewof the necrotic areas present in tumors and in view of the need totransport tumor tissue from a treatment or diagnosis center to alaboratory capable of performing the profiling analysis. By maintainingnon-necrotic portions of the tumor in a three-dimensional histoculture,the expression profile of the tumor in situ is effectively preserved.

DISCLOSURE OF THE INVENTION

The invention provides a method to prepare an mRNA librarycharacteristic of expression for use in profiling tissue, especiallytumor tissue, in order to characterize the nature of the tissue. In thecase of tumor analysis, this profile is helpful in designing treatment,especially in comparison with historical samples with similar expressionpatterns whose responsiveness to certain protocols is known. Other usesof characteristic expression profiles as related to particular tissuesources will be apparent to the skilled artisan.

Thus, in one aspect, the invention is related to a method to prepare RNAcharacteristic of a tissue expression which method comprises, afterculturing an intact tissue sample in sponge-gel three-dimensionalculture, extracting RNA from said culture.

The messenger RNA extracted can then be analyzed using any artrecognized technique, such as Northern blot. Preferably, however, theextracted mRNA is used as a template to prepare a cDNA library which canthen be analyzed using recognized array techniques, such as those basedon GeneChips. In other aspects, the invention is directed to mRNA, cDNAand cRNA libraries prepared by the method of the invention and tomethods to utilize these libraries for prognosis and treatmentselection.

MODES OF CARRYING OUT THE INVENTION

The present invention solves the problem of adequately preserving tissuesamples, especially tumor tissue samples, for expression profiling.Presently, biopsied samples are subject to RNA degradation andalteration in expression patterns in the interval between the biopsy andthe extraction of RNA for analysis. By maintaining the tissue intact inthree-dimensional histoculture, the accuracy of the expression profileis preserved and the degradation of RNA is minimized.

In the method of the invention, the tissue is biopsied usingconventional techniques, and then divided into intact portions of theapproximate dimension of 1 mm³. Some variation in sample size is, ofcourse, permitted and, for example, pieces in the range of 0.25 mm³-5mm³, 0.5-3 mm³, preferably 1-2 mm³ are used. The intact tissue piece isthen placed into a three-dimensional histoculture, typically bycombining the intact sample with a collagen sponge-gel, such as thosedescribed in Singh, B., et al., (supra) and multiple additional papers,reviewed, e.g., by Hoffman, R. M., et al., Int'l J. Oncol. (1992)1:467-474; Hoffman, R. M., in Encyclopedia of Life Sciences (2001)Nature Publishing Group, London, and generally known in the art in thepractice of HDRA™. The collagen-type sponge-gel useful in the inventionmethods are used as a support for the tissue and thus the dimension ofthe sponge-gel is substantially greater than the dimension of thefragment; typically, the surface area of the sponge-gel is roughly twicethe diameter of the intact tissue sample. The three-dimensional cultureis then maintained in suitable medium, such as the media described inthe attached publications. The culture is maintained for as long asnecessary to preserve the sample for RNA extraction.

RNA extraction is carried out using techniques standard in the art.

The tissue sample that is the source of the expression library istypically tumor tissue. Thus, the invention method is particularlyuseful in assessing expression profiles for tumors of the breast, lung,colon, liver, stomach, pancreas, prostate, head and neck, ovary, andbrain. This list is non-exhaustive, as any solid tumor or, indeed, anytissue may be used as the source of the library.

The extracted RNA is then analyzed according to the needs of theinvestigator. Northern blot techniques may be used, but additionalinformation can be obtained using commercially available expressionarrays, for example, expression arrays now available from Affymetrix.Approximately 15 μg of labeled cDNA is required. Typically, theextracted RNA is converted into cDNA by reverse transcription, mostgenerally by priming with an oligo-dT primer coupled to the T7 RNApolymerase promoter. The resulting single-stranded cDNA is converted todouble-stranded DNA which thus produces a template suitable for T7polymerase-driven in vitro transcription (IVT). Labeled nucleotides areincorporated during the in vivo transcription to permit later detectionof the resulting cRNA. The resulting transcription product, cRNA, isthen used to provide the profile as detected by DNA arrays supplied onchips.

In the latter determination, the labeled cRNA is fragmented by heatingin Mg++ containing buffer and combined in a hybridization cocktailcontaining salmon sperm DNA, BSA and spiked control RNA'S. Approximately250 μl of cocktail is applied to GeneChips™ and hybridized for 16 hoursat 50° C. as described in the Affymetrix Gene Chip Expression AnalysisTechnical Manual (2001). As there described, after hybridization, thechips are taken through high and low stringency washes followed bystaining with phycoerythrin-labeled streptavidin (molecular probes)antibody amplification with biotinylated anti-streptavidin antibody(Vector Labs) and an additional staining with phycoerythrin labeledstreptavidin. After further washing, the arrays are digitized in anAffymetrix scanner and the images evaluated using Microarray Suite 5™software.

The foregoing description is merely exemplary of the variety oftechniques that could be used to analyze the RNA extracted from theculture according to the method of the invention.

The data obtained from the determination of the components of thelibrary can be used effectively to predict outcomes for subjects fromwhom tumor tissue is removed and analyzed according to the inventionmethod, and can also be used to design protocols for treatment, as wellas to predict chemosensitivity.

1. A method to prepare an expression profile of a tissue which methodcomprises the step of extracting RNA from an intact sample of saidtissue cultured in a three-dimensional collagen sponge-gel culture. 2.The method of claim 1, which further includes subjecting said RNA toanalysis to obtain expression data.
 3. The method of claim 1, whichfurther includes converting the extracted RNA into cDNA.
 4. The methodof claim 3, which further includes preparing labeled cRNA from said cDNAand analyzing said cRNA using microarray analysis.
 5. The method ofclaim 1, wherein said tissue is tumor tissue.
 6. The method of claim 5,wherein said tumor is of the breast, lung, colon, liver, stomach,pancreas, prostate, head, neck, ovary, or brain.
 7. An RNA expressionlibrary prepared by the method of claim
 1. 8. A cDNA expression libraryprepared by the method of claim
 3. 9. A cRNA expression library preparedby the method of claim
 4. 10. The method of claim 2, which furtherincludes preparing a prognosis based on said data.